FIGURE 3.

Three separate model schemes of long-term culturing/aging of Placozoa. Mode 1—optimized conditions; Mode 2—low and Mode 3—high densities of the green alga mat (Tetraselmis marina) or rice grains; see text for details. Mode 1: Optimized culture conditions with dispersal animals and their moderate concentration on feeding substrates. The middle diagram shows examples of exploratory and feeding locomotion for five animals (numbers) within 30 min. The right diagram shows the representative dynamics of the population growth rate (PGR) for all haplotypes (H1, H2, H4, and H13). 3 separate dishes were used for each haplotype (e.g., H4_1, H4_2, H4_3, etc.), starting with 10 animals per dish. All datasets were normalized to absorb the variation between columns for all four haplotypes of Placozoa. Under these conditions, animals steadily increase their body surface area and have vegetative (non-sexual) reproduction by fission. Mode 2: Low-density substrate. Limit of food source led to decreasing of animal sizes and numbers of animals in culture dishes. Mode 3: High-density substrate. There is both increasing in animal sizes and the aggregation of 2–15 individuals around rice grains or on the dense algal mat. The heat diagram on the right shows the predominant occurrence of aggregates compared to Mode 1 (no aggregates were observed on low-density substrates in Mode 2). H4 expressed the same behavior patterns on the walls of 20L aquarium, where individuals within the aggregate could move together (1-2-3, arrows). However, most animals stay at the substrate (central diagram) with significantly reduced overall locomotion during the feeding, as indicated in the right middle diagram. Each set of video images (Mode 1–3) was analyzed using ImageJ (NIH), calculating for velocity, animal area, and perimeter (n = 3–6), as was reported elsewhere (Romanova et al., 2020a). Difference between locomotion in Modes 1 and 3: p-value is 0.003002; between Modes 2 and 3: p-value is 0.000072 (unpaired Student’s test). Scale bars: for individual animals—200 μm; for the aggregate in Mode 3—1 mm; for all locomotory tracks—200 µm.